Ceacam1 based point-of-care cancer diagnostic

ABSTRACT

The present technology regards the utilization of CAECAM1 as a biomarker for cancer. In particular the present invention regards the detection and measurement of soluble CAECAM1 levels for the detection and diagnosis of melanoma.

CROSS-REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCE

The present application is a continuation of U.S. patent applicationSer. No. 12/265,469, filed Nov. 5, 2008, which is related to and claimspriority from U.S. Provisional Patent Application No. 60/985,484, filedNov. 5, 2007, and is titled “CEACAM1 BASED POINT-OF-CARE CANCERDIAGNOSTIC,” the contents of which are herein incorporated by referencein their entirety.

FIELD OF THE INVENTION

This invention relates generally to the field of cancer detection, andmore specifically to the utilization of CAECAM1 as a biomarker for thepoint-of-care diagnosis of cancer.

BACKGROUND OF THE INVENTION

Cancer is a term for diseases in which abnormal cells divide withoutcontrol. Cancer cells can invade nearby tissues and can spread to otherparts of the body through the blood and lymph systems. There are severaltypes of cancer, including without limitation, carcinoma which is cancerthat begins in the skin or in tissues that line or cover internalorgans. Sarcoma is cancer that begins in bone, cartilage, fat, muscle,blood vessels, or other connective or supportive tissue. Leukemia iscancer that starts in blood-forming tissue such as the bone marrow, andcauses large numbers of abnormal blood cells to be produced and enterthe blood. Lymphoma and multiple myeloma are cancers that begin in thecells of the immune system. Central nervous system cancers are cancersthat begin in the tissues of the brain and spinal cord.

The human carcinoembryonic Ag (CEA)3 protein family encompasses severalproteins with different biochemical features. These proteins are encodedby 29 genes tandemly arranged on chromosome 19q13.2. CEA family geneshave been classified into two major subfamilies, the CEA cell adhesionmolecule (CEACAM) and the pregnancy-specific glycoprotein subgroups. TheCEACAM proteins, which are part of the larger Ig superfamily, includeCEACAM1, -3, -4, -5, -6, and -8. They share a common basic structure ofsequentially ordered different Ig-like domain(s) and are able tointeract with each other. For example, it has been reported that variousCEACAM proteins, such as CEACAM1 or CEACAM5, exhibit both homophilic andheterophilic interactions.

CEACAM1 (CD66a), a transmembrane protein and member of thecarcinoembryonic Ags family (belongs to IgSF), contains two ITIMsequences located within its cytosolic tail. CEACAM1 interacts withother known CD66 proteins (both homophilic and heterophilicinteractions), including CD66a, CD66c, and CD66e proteins. It isexpressed on a wide spectrum of cells, ranging from epithelial tohemopoietic origin. Among CD66 proteins tested, only the CD66a proteinis expressed on the surface of activated CD16-negative NK cells.

Point-of-care testing refers to a medical test which is carried out atsites other than a central laboratory, and comprises testing at or nearthe site of patient care. The value added by Point-of-Care Diagnosticsis significant, and includes for example: improved patient outcomesattributable to immediate on-site actionable healthcare resulting fromimmediate test results (i.e.—reduced time for start of treatment);access to areas lacking clinical laboratory infrastructures (i.e.—ruralareas, disaster areas, and developing countries); and avoidance ofsample identification and sample transport problems.

BRIEF SUMMARY OF THE INVENTION

The present technology generally regards methods for diagnosing cancercomprising the detection of soluble CAECAM1 in a biological sample. Inone particular aspect, this technology relates to animmunochromatographic strip for the point-of-care detection of solubleCEACAM1 in a biological sample for the detection and prognosis ofmelanoma.

The present technology further regards a diagnostic device forpoint-of-care detection of cancer comprising a matrix material having anapplication zone wherein a test fluid is applied, the test fluid movingby capillary action into a reaction zone comprising a mobile phasehaving a first CEACAM1 binding element (i.e.—a first CAECAM1 antibody orfragment thereof) conjugated to a detectable moiety, the test fluid thenmoving by capillary action through the reaction zone into one or morecapture zones having a stationary phase comprising a second CEACAM1binding element (i.e.—a second CAECAM1 antibody or fragment thereof),the detection of CEACAM1 in the test sample being dependent upon thepresence of said detectable moiety within the CEACAM1 capture zone.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. Presentation of data showing that soluble CEACAM1 is increasedin melanoma patients.

FIG. 2. Schematic representation of an immunochromatographic strip forthe point-of-care detection of CEACAM1 in a biological sample.

FIG. 3. Schematic representation of one embodiment of a lateral-flowpoint-of-care rapid detection system, for the detection and measurementof CEACAM1 in a test sample.

DETAILED DESCRIPTION OF THE INVENTION

The present technology generally regards the use of soluble CEACAM1 forthe diagnosis and for predicting the prognosis of cancer, including forexample melanoma. The present technology also comprises in part aimmunochromatographic strip for the point-of-care detection of CAECAM1in a biological sample.

CEACAM1 appears as a soluble molecule in the peripheral blood. Thepresent technology regards the diagnosis of cancer comprising thedetection of soluble CAECAM1 in a biological sample. The data shown inTable 1 and presented in FIG. 1 demonstrates that soluble CEACAM1 levelsfunction as a biomarker for at least one type of cancer, namely melanoma(a form of skin cancer that begins in melanocytes (the cells that makethe pigment melanin). As shown in FIG. 1, soluble CEACAM1 is increasedin melanoma patients (LR (Low Risk); HR (High Risk); Met (Metastaticpatients); healthy donors.). Soluble CEACAM1 is quantified using aCEACAM1 specific sandwich ELISA. For example, the specific anti-CEACAM15F4 mAb can be used as capturing antibody. For detection, biotinylatedKat4c mAb can be used, followed by streptavidin-horseradish peroxidase(Jackson ImmunoResearch). Biotinylation of the Kat4c mAb can beperformed with Sulfo-NHS-SS-Biotin (Pierce) according to themanufacturer's instructions. The quantification can then be calculatedaccording to standard samples of CEACAM1-Ig fusion proteins.

FIG. 2 is an schematic illustration of one aspect of the presenttechnology. Referenced generally as 1 is a immunochromatographic stripcomprising a matrix material 2 having an application (or sample loading)zone 3, a reaction zone 4, a CEACAM1 capture zone 5, a control analytecapture zone 6, and an optional wicking pad 7.

The test fluid (and any CEACAM1 suspended or dissolved therein) is firstbrought into contacted with or applied to the application zone 3 of theimmunochromatographic strip 1. The immunochromatographic strip 1comprises a matrix material 2 which facilitates the test fluid moving bycapillarity action from the application zone 3 through the reaction zone4 and past the capture zones (CEACAM1 capture zone 5 and control analytecapture zone 6) to an optional wicking pad 7. Any detectable signal inthe capture zones will reveal the presence of analyte (CEACAM1 orcontrol).

The matrix material comprises a membrane material, for examplenitrocellulose membrane, cellulose acetate membrane, glass-fibermembrane, or any combinations or derivatives thereof. The matrixmaterial is porous, and any test liquid or sample applied to the matrixmaterial at application zone 3 can move by capillary into the reactionzone 4 and continue to and/or past the capture zones 4 and 5.

The reaction zone 4 contains a mobile phase comprising a first CEACAM1specific antibody (or fragment thereof) conjugated to a detectablemoiety (e.g.—radioactive isotopes, enzymes, dyes, fluorescent dyes, dyedmicrospheres, affinity tag, or any combination or derivative thereof).The reaction zone 4 will also comprise, in mobile phase, a first controlanalyte specific antibody conjugated to a detectable moiety. When thetest sample (and any CEACAM) moves by capillary action out of theapplication zone 3 and into the reaction zone 4, an antigen-antibodyreaction ensues resulting in any CEACAM1 becoming bound (in mobilephase) by a first CEACAM1 specific antibody (or fragment thereof)conjugated to a detectable moiety. See FIG. 3.

The antigen (CEACAM1)-antibody reaction product formed within thereaction zone then migrates with the test liquid into one or moredownstream capture zones having immobilized or stationary antibody. TheCEACAM1 capture zone 5 comprises an immobilized or stationary phasesecond CEACAM1 binding element (e.g.—a second CAECAM1 antibody orfragment thereof) or CEACAM1 capture agent (e.g.—CEACAM1 captureantibody). The control analyte capture zone 6 also comprises animmobilized or stationary phase second control analyte binding element(e.g.—a second control analyte antibody or fragment thereof) or controlanalyte capture agent (e.g.—control analyte capture antibody). See FIG.3.

As a result of capillary flow from the application zone 3 through thereaction zone 4 and into the capture zones 5 and 6, a concentration ofthe detectable moiety builds within the capture zones, and theconcentration of CEACAM1 in capture zone 5 (and control analyte(s) incapture zone 6) can then be assessed qualitatively or quantitativelywith a suitable measuring instrument, or if dyes that are adsorbent inthe visible range are used as the detectable moiety, can be perceivedwith the naked eye (FIG. 3).

The detection moiety can be radioactive isotopes, enzymes, dyes,fluorescent dyes, dyed microspheres, affinity tag, or any combination orderivative thereof.

The CEACAM1 binding agent (or capture agent) include without limitationan antibody (or fragment thereof) specific for CEACAM1. The antibody canbe either polyclonal or monoclonal. Exemplar CEACAM1 specific antibodiesinclude: Mouse monoclonal [29H2] to CEACAM1; Mouse monoclonal [GM8G5] toCEACAM1 (GM8G5 recognizes the Human CEACAM1 A2 domain); CEACAM1 antibodynumber 2037.00.02 (from Strategic Diagnostics Inc) binding CEACAM1 aminoacids 35-134; the CEACAM1-specific antibody 4D1/C2; anti-CEACAM1 5F4mAb; anti-CEACAM1 Kat4c mAb; or any combination or derivative thereof.The CEACAM1 binding agent can also be a member of the CEA proteinfamily.

What is claimed is:
 1. A device for the point-of-care detection ofsoluble CAECAM1 in a biological sample, the device comprising animmunochromatographic strip for the point-of-care detection of CEACAM1in a biological sample.
 2. The device of claim 1, wherein the biologicalsample is blood, blood serum, or urine.
 3. The device of claim 1,wherein said immunochromatographic strip comprises a porous matrixhaving a sample application zone, a reaction zone, and a capture zone incommunication with each other, said porous matrix supporting thecapilary movement of said biological sample.
 4. The device of claim 3,wherein the reaction zone comprises a mobile phase comprising a CEACAM1specific antibody or fragment thereof conjugated to a detectable moiety5. The device of claim 4, wherein said detectable moiety comprises aradioactive isotope, an enzyme, a fluorescent dye, a dyed microsphere,an affinity tag, or any combination or derivative thereof.